PROJECT SUMMARY/ABSTRACT Cre-loxP-based cell type-specific loss of function (LOF) studies have expanded our understanding of skeletal biology and identified cellular and molecular mechanisms underpinning skeletal pathologies. However, extensive use of the Cre-loxP system has revealed important shortcomings of this methodology such as its activity in off-target cell types. Therefore, there is a significant need for new methodologies to perform cell type-specific LOF studies. CRISPR inhibition/interference (CRISPRi) is a derivative of the CRISPR-Cas9 system, developed to suppress target genes, and constitutes an alternative method to perform LOF. In this methodology, the two endonuclease domains of Cas9 protein are mutated to prevent Cas9's ability to excise DNA. This dead Cas9 (dCas9) is fused to transcriptional suppressor domains, such as KRAB, to produce the repressive fusion protein dCas9::KRAB. Once dCas9::KRAB protein is directed to transcriptional start sites (TSS) of target genes via a single guide RNA (sgRNA), this fusion protein suppresses expression of target genes. To test whether CRISPRi can be utilized in vivo, we recently produced a murine model for global CRISPRi of Tnfsfs11. Different founder lines of this murine model termed Tnfsf11gCRISPR exhibited dose-dependent suppression of RANKL. Moreover, the phenotype of Tnfsf11gCRISPR founder lines that expressed high levels of dCas9::KRAB was comparable to that of RANKL null mice. Based on the success of gCRISPRi and the observed in vivo dose-dependency of CRISPRi, we hypothesize that CRISPRi-based transgenes can be used to perform loss of function studies in mice that are more specific than those performed with the Cre-loxP system but are as effective. This project will produce osteocyte-specific CRISPRi models and compare the specificity and efficacy of cell type- specific CRISPRi compared to the Cre-loxP system. Specifically, in the first aim we will test whether a Dmp1 promoter driven CRISPRi transgene will suppress a target gene more specifically than a Dmp1-Cre transgene. In the second aim, we will compare the efficacies of CRISPRi and Cre-loxP systems. For this purpose, we will perform osteocyte-specific LOF of RANKL using CRISPRi and Cre-loxP methodologies and compare the resultant phenotypes as well as the decrease in RANKL levels. Establishing the specificity and effectiveness of in vivo CRISPRi for cell type-specific LOF studies also lays the ground work for future studies aimed at utilization of other advantages of the CRISPRi approach, such as suppression of multiple target genes with one CRISPRi transgene.